Luminescence spectral properties of CdS nanoparticles

ABSTRACT

The steady state and time resolved luminescence spectral properties of two types of novel CdS nanoparticles and nanoparticles are described. CdS nanoparticles formed in the presence of an amine-terminated dendrimer show blue emission. The emission wavelength of these nanoparticles depended on the excitation wavelength. The CdS/dendrimer nanoparticles display polarized emission with the anisotropy rising progressively from 340 to 420 nm excitation, reaching a maximal anisotropy value in excess of 0.3. A new constant positive polarized emission from luminescent nanoparticles is also described. Polyphosphate-stabilized CdS nanoparticles are described that display a longer wavelength red emission maximum than bulk CdS and display a zero anisotropy for all excitation wavelengths. Both nanoparticles display strongly heterogeneous intensity decays with mean decay times of 93 ns and 10 μs for the blue and red emitting particles, respectively. Both types of nanoparticles were several times more photostable upon continous illumination than fluorescein. In spite of the long decay times the nanoparticles are mostly insensitive to dissolved oxygen but are quenched by iodide. These nanoparticles can provide a new class of luminophores for use in chemical sensing, DNA sequencing, high throughput screening and other applications.

This patent application claims priority to U.S. patent application60/118,904, filed on Feb. 5, 1999, and to International patentapplication PCT/US00/02954 filed on Feb. 4, 2000, copies of which areincorporated by reference.

The United States Government may have rights to this invention pursuantto the National Institute of Health (NIH), National Center for ResearchResources, Grant No. RR-08119.

FIELD OF THE INVENTION

This invention relates to nanoparticle cadmium sulfide (CdS) fluorescentprobes. Preferably, this invention relates to CdS nanoparticles formedin the presence of an amine-terminated dendrimer and/orpolyphosphate-stabilized CdS particles both with average diameters orother critical dimensions (CDs) of several nanometers (nm).

BACKGROUND

There is presently widespread interest in the physical and opticalproperties of semiconductor particles with average diameters or CdSmeasured in nanometers. These particles are often called nanoparticlesor quantum dots. The optical properties of such particles depends ontheir size [Martin, C. R.; Mitchell, D. T., Anal. Chem. (1998)322A-327A].

Such particles display optical and physical properties which areintermediate between those of the bulk material and those of theisolated molecules. For example, the optical absorption of bulk CdSetypically extends to 690 nm. The longest absorption band shifts to 530nm for CdSe nanoparticles with 4 nm average diameters [Bawendi, M. G.;et al., Annu. Rev. Phys. Chem. (1990) 41, 477-496].

Sizes of nanoparticies are usually measured by average diameters ofequivalent spherical particles. For particles that are not at leastapproximately spherical, the smallest dimension (called criticaldimension or CD) is often used. In nanoparticles a large percentage ofthe atoms are at the surface, rather than in the bulk phase.Consequently, the chemical and physical properties of the material, suchas the melting point or phase transition temperature, are affected bythe particle size. Semiconductor nanoparticles can be made from a widevariety of materials including, but not limited to CdS, ZnS, Cd₃P₂, PbS,TiO₂, ZnO, CdSe, silicon, porous silicon, oxidized silicon, andGa/InN/GaN.

Semiconductor nanoparticles frequently display photoluminescence andsometimes electroluminescence. For example see Dabbousi, B. O., et al.,Appl. Phys. Lett. (1995) 66(11), 1316-1318; Colvin, V. L., et al.,Nature, (1994) 370, 354-357; Zhang, L., et al., J. Phys. Chem. B. (1997)101 (35), 874-6878; Artemyev, M. V., et al, J. Appl. Phys., (1997)81(10), 6975-6977; Huang, J., et al., Appl. Phys. Lett. (1997) 70(18),2335-2337; and Artemyev, M. V., et al., J. Crys. Growth, (1988) 184/185,374-376. Additionally, some nanoparticles can form self-assembledarrays.

Nanoparticles are being extensively studied for use in optoelectronicdisplays. Photophysical studies of nanoparticles have been hindered bythe lack of reproducible preparations of homogeneous size. The particlesize frequently changes with time following preparation. Particlesurface is coated with another semiconductor or other chemical speciesto stabilize the particle [Correa-Duarte, M. A., et al., Chem. Phys.Letts. (1998) 286, 497-501; Hines, M. A., et al., J. Phys. Chem. (1996)100, 468-471; and Sooklal, K., et al., J. Phys. Chem. (1996) 100,4551-4555].

There are several examples of fluorescing cadmium sulfide nanoparticles.Tata, et al. use emulsions [Tata, M., et al., Colloids and Surfaces,127, 39 (1997)]. Fluorescence of CdS nanocrystals have been observed bylow temperature microscopy. Blanton, et al. show fluorescence from 5.5nm diameter CdS nanocrystals with excitation of 800 nm and emissioncentered around 486 nm [Blanton, S., et al., Chem. Phys. Letts., 229,317 (1994)]. Tittel, et al. noticed fluorescence of CdS nanocrystals bylow temperature confocal microscopy [Tittel, J., et al., J. Phys. Chem.B, 101(16) (1997) 3013-3016].

A 64 branch poly(propylene imine) dendritic box can trap a Rose Bengalmolecule (i.e., a polyhalogenated tetracyclic carboxylic acid dye) toallow it to strongly fluoresce since it is isolated from surroundingquenching molecules and solvents [Meijer, et al., Polym. Mater. Sci.Eng., (1995) 73, 123].

While the absorption and emission spectra of nanoparticles have beenwidely studied, the scope of these measurements were typically limitedto using the optical spectra to determine the average size of theparticles. There have been relatively few studies of the time-resolvedphotophysical properties of these particles.

The emission from silicon nanoparticles has been reported as unpolarized[Brus, L. E., et al., J. Am. Chem. Soc. (1995) 117, 2915-2922] orpolarized [Andrianov, A. V., et al., JETP Lett. (1993) 58, 427-430;Kovalev, D., et al., Phys. Rev. Letts. (1997) 79(1), 119-122; and Koch,F., et al., J. Luminesc., (1996) 70, 320-332]. Polarized emission hasalso been reported for CdSe [Chamarro, M., et al., Jpn. J. Appl. Phys.(1995) 34, 12-14; and Bawendi, M. G., et al., J. Chem. Phys. (1992)96(2), 946-954]. However, in these cases the polarization is eithernegative or becomes negative in a manner suggesting a process occurringwithin the nanoparticle. Such behavior would not be useful for afluorescence probe for which the polarization is expected to depend onrotational diffusion.

The increasing availability of homogeneous sized nanoparticles suggestsmore detailed studies of their photophysical properties, which in turncould allow their use as biochemical probes. The first reports of suchparticles as cellular labels have just appeared [Bruchez, M., et al.,Science (1998) 281, 2013-2016; and Chan, W., et al., Science (1998) 281,2016-2018]. CdS particles have also been synthesized which bind DNA anddisplay spectral changes upon DNA binding [Mahtab, R., et al., J. Am.Chem. Soc. (1996) 118, 7028-7032; and Murphy, C. J., et al., Proc.Materials Res. Soc. (1997) 452, 597-600].

U.S. Pat. No. 5,938,934 to Balogh et al., describes use of dendrimers ashosts for many materials including semiconductors. However thenanoparticles are somewhat large for use as a probe based on size. Onlyexample 15 discloses cadmiums sulfide. However dangerous sulfide gas isused over prolonged periods of time.

SUMMARY

This invention describes fabrication methods, spectroscopy, probes andother applications for semiconductor nanoparticles. The preferredembodiments are two types of cadmium sulfide (CdS) nanoparticles. CdSnanoparticles formed in the presence of an amine-terminated dendrimershow blue emission. The emission wavelength of these nanoparticlesdepends on the excitation wavelength. These CdS/dendrimer nanoparticlesdisplay a new constant positive polarized blue emission.Polyphosphate-stabilized CdS nanoparticles are described that display alonger wavelength red emission maximum than bulk CdS and display a zeroanisotropy for all excitation wavelengths. Both nanoparticles displaystrongly heterogeneous intensity decays with mean decay times of 93 nsand 10 μs for the blue and red emitting particles, respectively. Bothtypes of nanoparticles were several times more photostable uponcontinuous illumination than fluorescein. In spite of the long decaytimes the nanoparticles are mostly insensitive to dissolved oxygen butare quenched by iodide. These nanoparticles can provide a new class ofluminophores for use in chemical sensing, DNA sequencing, highthroughput screening, fluorescence polarization immunoassays, time-gatedimmunoassays, time-resolved immunoassays, enzyme-linked immunosorbentassay (ELISA) assays, filtration testing, and targeted tagging and otherapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows absorption and emission spectra for the blue emittingCdS/dendrimer nanoparticle in methanol at room temperature. Theexcitation spectrum of this nanoparticles overlaps with the absorptionspectrum. Also shown are the excitation and emission anisotropy spectra,also in methanol at room temperature.

FIG. 2 shows photostability tests of the CdS/dendrimer andpolyphosphate-stabilized (PPS) nanoparticles. The sample was containedin a standard 1 cm×cm (4 mL) cuvette. The incident power was 30 mW at405 nm from a frequency-doubled Ti:Sapphire laser, 80 MHz, 200 fs, whichwas focused with a 2 cm focal length lens. Also shown is the intensityfrom fluorescein, pH 8, under comparable conditions. When illuminatedwith the output of a 450 W xenon lamp (385 nm for blue and 405 nm forred nanoparticles) there was no observable photobleaching.

FIG. 3 shows emission spectra of the CdS/dendrimer composite fordifferent excitation wavelengths. Also shown as the dashed line is thetransmission profile of the filter used for the time-resolvedmeasurements.

FIG. 4 shows a frequency-domain intensity decay of the CdS/dendrimernanoparticle for excitation at 395 nm (top) and 325 nm (bottom). Thissolid line shows the best three decay time fit to the data.

FIG. 5 shows time-dependent intensity decays of the nanoparticlesreconstructed from the frequency-domain data (Tables I and II).

FIG. 6 shows a frequency-domain anisotropy decay of the CdS/dendrimernanoparticle for excitation at 395 nm, at room temperature in methanol.

FIG. 7 shows absorption (A) and emission (F) spectra of the CdS/PPSnanoparticles. In this case the emission spectra were found to beindependent of excitation wavelengths from 325 to 450 nm. The dashedline shows the transmission of the filter used to record thetime-resolved data.

FIG. 8 shows excitation anisotropy spectra of the CdS/PPS nanoparticlesin 80% glycerol at −60° C. (dots). Also shown are the temperaturedependent spectra in 80% glycerol.

FIG. 9 shows a frequency-domain intensity decay of the CdS/PPSnanoparticles for excitation at 442 nm (top) and 325 nm (bottom). Thesolid lines show the best three decay time fits to the data.

FIG. 10 shows the effect of oxygen on the emission spectra of theCdS/dendrimer and CdS/PPS nanoparticles.

FIG. 11 shows the effect of acrylamide and iodide on the emissionspectra of the CdS/dendrimer and CdS/PPS nanoparticles.

FIG. 12 shows intensity decays of the CdS/dendrimer (top) and CdS/PPSnanoparticles (bottom) in the absence and presence of 0.2 M acrylamideor 0.2 M iodide. These measurements were done independently of thosepresented in FIGS. 4 and 9. For the CdS/dendrimer nanoparticle (toppanel) the recovered average lifetimes (τ=Σf_(i)τ_(i)) are: 106.0 ns fornot quenched (), 73.7 ns in presence of 0.2 M acrylamide (∘) and 36.7ns in presence of 0.2 M KI (▾). For the CdS/PPS nanoparticles (lowerpanel) average lifetimes are 9.80 μs for not quenched (), 8.45 μs inpresence of 0.2 M acrylamide (not shown), and 4.09 μs in presence 0.2 MKI (▾).

DETAILED DESCRIPTION

This invention describes detailed studies of the steady state andtime-resolved emission semiconductor nanoparticles. The preferredembodiments are two types of stabilized CdS particles. The first type ofCdS nanoparticles were fabricated in the presence of a dendrimer anddisplay blue emission. The second type of CdS particles were stabilizedwith polyphosphate and display red emission.

Semiconductor nanoparticles with fluoresce and/or luminesce moreintensely and often at wavelengths shifted from their bulk counterparts.The nanoparticles of the present invention luminesce most strongly whenthey have average diameters and/or critical dimensions less than 5 nm.The nanoparticles of the present invention have a very narrowdistribution of diameters and/or critical dimensions. In the preferredmode of this invention, at least 90% of a nanoparticle powder hascritical dimensions of no more than +/−15% from the average diameterand/or critical dimension of the powder. This narrow particle sizedistribution is extremely important for maximizing emission intensityand other fluorescent properties.

The semiconductor nanoparticles of the present invention may be only onesemiconductor, composites of several materials in each nanoparticle,and/or mixtures of different nanoparticles (e.g., powders, agglomerates,and/or aggregates). The individual nanoparticles can be uncoated,coated, partially coated, attached to a molecule, and/or trapped in ananoscopic volumetric area. In one contemplated example, a semiconductornanoparticle is coated with another semiconductor. The coatingpreferably has a higher bandgap than the core nanoparticle. In anothercontemplated example, electrically non-conductive coatings or anchormolecules control the size and spacing of the semiconductivenanoparticles. Coatings can also be used to protect the corenanoparticle from other effects such as, but not limited to, certainwavelengths, oxidation, quenching, size changes, size distributionbroadening, and electronic conductivity. There may be more than onecoating layer and/or material.

The nanoparticles of the present invention have at several importantimprovements. First, the dendrimer-based and other types oftemplate-based nanoparticles show polarized emission. Polarizationoffers many advantages and an additional variable over prior fluorescentnanoparticle spectroscopy. Second, the nanoparticles of the presentinvention are very resistant to quenching by oxygen or other dissolvedspecies. This important advance avoids the quenching problems thatplague much of fluorescence spectroscopy. Third, the nanoparticles ofthe present invention have long wavelength emission. Emissionwavelengths of above 500 nm possible with the present invention areespecially suitable for biological sensing and minimize autofluorescentnoise. Fourth, the nanoparticles of the present invention have longlifetimes. Lifetimes of 30 ns to well over 100 ns are possible with thisinvention even in the presence of fluorescence quenchers. Long lifetimesallow use of smaller and less expensive spectrometers, sensors anddetectors. The combination of long lifetimes with long fluorescencedecay times are particularly valuable.

This invention's preferred mode describes solution phase nanofabricationof semiconductor nanoparticles. Solution phase nanofabrication is muchless expensive than most types of nanofabrication using vacuum systems,electrochemistry, special ball mills, electric arcs, gas phasechemistry, etc. This invention's nanoparticles can be made in bulk orwithin a template such as, but not limited to, a dendrimer membrane ordendrimer-modified optical fiber. This invention avoids the use ofdangerous and expensive reactive gases such as sulfide gas.

CdS/Dendrimer Nanoparticle

FIG. 1 shows the absorption and emission spectra of the CdS/dendrimerparticles. There is a substantial Stokes' shift from 330 to 480 nm. Sucha large Stokes' shift is a favorable property because the emission ofthe nanoparticles will be observable without homo-energy transferbetween the particles. Also, because of the substantial shift it shouldbe relatively easy to eliminate scattered light from the detected signalby optical filtering. The term nanoparticle in this invention is meantto include nanocomposites, clusters of nanoparticles, agglomerates ofgenerally electrically isolated nanoparticles and surface-modifiednanoparticles as well as single material particles.

The emission intensity of the blue nanoparticles is relatively strong.The relative quantum yield is estimated by comparing the fluorescenceintensity with that of a fluorophore of known quantum yield, and anequivalent optical density at the excitation wavelength of 350 nm. Asolution of coumarin 1 in ethanol with a reported quantum yield of 0.73was used as a quantum yield standard. This comparison yields an apparentor a relative quantum yield of 0.097. This value is not a molecularquantum yield because there is no consideration of the molarconcentration of the nanoparticles. However, this value does indicatethe relative brightness of the particles as compared to a knownfluorophore. This value is somewhat lower than the previously reportedquantum yield of approximately 0.17 [Murphy, C. J., Brauns, E. B., andGearheart, L. (1997), Quantum dots as inorganic DNA-binding proteins,Proc. Materials Res. Soc. 452, 597-600]. It is possible that the quantumyields differ for different preparations of the nanoparticles.

For use as a luminescent probe the signal from the nanoparticles must bestable with continual illumination. The emission intensities and/oremission spectra of nanoparticles occasionally depend on illumination.In contrast, the CdS/dendrimer particles appear to be reasonably stableand about two-fold more stable than fluorescein (FIG. 2). In thesestability tests the fluorescein and nanoparticles were illuminated withthe focused output of a frequency-doubled Ti:Sapphire laser. No changesin the emission intensity of the nanoparticles were found whenilluminated with the output of a 450W xenon lamp and monochromator.

For use as a biophysical probe of hydrodynamics a luminophore mustdisplay polarized emission. Since most nanoparticles are thought to bespherical, the emission is not expected to display any usefulpolarization. Importantly, the CdS/dendrimer nanoparticles of thepresent invention display high anisotropy (FIG. 1). This anisotropyincreases progressively as the excitation wavelength increases acrossthe long wavelengths side of the emission, from 350 to 430 nm. Theemission anisotropy is relatively constant across the emission spectra.These properties, and the fact that the anisotropy does not exceed theusual limit of 0.4, suggest that the emission is due to a transitiondipole similar to that found in excited organic molecules. The high andnon-zero anisotropy also suggests that the excited state dipole isoriented within a fixed direction within the nanoparticles.

A fixed direction for the electronic transition suggests the presence ofsome molecular features which define a preferred direction for thetransition moment. While most nanoparticles are thought to be spherical,the shape of the CdS inside of the CdS/dendrimer nanoparticle is notknown. Electron micrographs show that the particles and dendrimers existas larger aggregates rather than as isolated species. Unfortunately, thepresence of aggregates prevented determination of the particle shape.Our observation of a large non-zero anisotropy for these particlessuggests an elongated shape for the quantum-confined state. This is thefirst constant positive polarized emission from CdS nanoparticles. Theresults in FIG. 2 suggest that CdS/dendrimer nanoparticles can serve ashydrodynamic probes for rotational motions on the 50 to 400 ns timescale(see FIG. 4 below).

If the particle preparation has a single particle size, the emissionspectra are expected to be independent of excitation wavelength. Hencewe recorded the emission spectra for the CdS/dendrimer particles for arange of excitation wavelengths (FIG. 3). Longer excitation wavelengthsresults in a progressive shift of the emission spectra to longerwavelengths. This effect is reminiscent of the well-known red edgeexcitation shift observed for organic fluorophores in polar solvents.However, the molecular origin of the shift seen in FIG. 3 is different.In this case the shifts are probably due to the wavelength-dependentexcitation of a selected sub-population of the particles at eachwavelength. In particular, longer excitation wavelengths probablyresults in excitation of larger particles with a longer wavelengthemission maximum. Hence this particular preparation of CdS/dendrimerparticles appears to contain a range of particle sizes. However, wecannot presently exclude other explanations for the wavelength-dependentspectra seen in FIG. 3.

We examined the time-resolved intensity decay of the CdS/dendrimerparticles using the frequency-domain (FD) method [J. R. Lakowicz and I.Gryczynski, Topic in Fluorescence Spectroscopy, Vol I, Techniques,Plenum Press, New York, pp 293-355]. The frequency responses were foundto be complex (FIG. 4), indicating a number of widely spaced decaytimes. The FD data could not be fit to a single or double decay timemodel (Table I). Three decay times were needed for a reasonable fit tothe data, with decay times ranging from 3.1 to 170 ns. The mean decaytime is near 117 ns. There seems to be a modest effect of excitationwavelength. The mean decay time decreases from 117 ns for excitation at395 nm to 93 ns for excitation at 325 nm. Such long decay times are avaluable property for a luminescent probe, particularly one which can beused as an anisotropy probe. The long decay time allows the anisotropyto be sensitive to motions on a timescale comparable to the meanlifetime. Hence, it is envisioned to use these nanoparticles as probesfor the dynamics of large macro molecular structures, or even as modelproteins since the nanoparticle size is comparable to the diameter ofmany proteins.

To better visualize the intensity decays, the parameters (α_(i) andτ_(i)) recovered from the least-squares analysis in Table I were used toreconstruct the time-dependent intensity decays (FIG. 5). The intensityis multi- or non-exponential at early times (insert), but does notdisplay any long-lived microsecond components. While the intensity decaycould be fit to three decay times, it is possible that the actual decayis more complex, and might be more accurately represented as adistribution of decay times.

In the frequency-domain anisotropy decay of the CdS/dendrimer particles(FIG. 6), the differential polarized phase angles are rather low, withthe largest phase angles centered near 1.0 MHz, suggesting rather longcorrelation times for the particles. Least squares analysis of the FDanisotropy data revealed a correlation time near 2.4 μs (Table I). Sucha long correlation time is consistent with the observation that the CdSnanoparticles are aggregated with the dendrimers, or somehow present ina composite structure. Much shorter correlation times would be expectedfor particles with sizes near 2 nm that would be consistent with theoptical properties. The time-zero anisotropy recovered from the FDanisotropy data is consistent with that expected from the excitationanisotropy spectra and the excitation wavelength. This agreementsuggests that the anisotropy of these particles decays due to overallrotational motion, and not due to internal electronic properties of theparticles. It is envisioned that these nanoparticles (especially whennot aggregated) are useful as analogues of proteins or othermacromolecules, and as internal cellular markers which could report therate of rotational diffusion.

Dendrimers are macromolecules such as poly(amidoamine-organosilicon)containing hydrophilic and hydrophobic nanoscopic domains. The dendrimerhave a dense star architecture which is a macromolecular structure withchains that branch from a central initiator core. Dendrimers have narrowmolecular weight distributions with specific sizes and shapes. Thedendrimers grow larger with each generation. For example, a generation 4dendrimer is smaller than a generation 5 dendrimer. Dendrimers also havehighly functional and accessible terminal surfaces. In the preferredembodiment of this invention, this terminal surface has amine which canbind cadmium. In the present invention, each dendrimer preferably holdsa plurality of cadmium sulfide or other semiconductor nanoparticles.Creating semiconductor nanoparticles in dendrimer-based nanoscopicmolecular sponges and dendrimer-based network materials (e.g.,elastomers, plastomer, coatings, films and membranes) are alsoenvisioned. The present invention can any non-conductive system havingnanoscopic domains capable of binding a semiconductor. Envisionedexamples include, but are not limited to, dendrimers, star polymers,self-assembling polymers, and zeolites.

Polyphosphate-Stabilized CdS Nanoparticles

Other CdS nanoparticles in this invention, called CdS/PPS, have surfacesstabilized with polyphosphate (PPS). Absorption and emission spectra ofthese particles are shown in FIG. 7. Compared to the CdS/dendrimernanoparticles, these stabilized nanoparticles absorbs and emit at muchlonger wavelengths. Their average diameter was estimated to be 4 nm±15%by transmission electron microscopy. The spectra and intensities werefound to be stable with prolonged illumination and at least four-foldmore stable than fluorescein (FIG. 2). The emission intensity of thesered-emitting particles is considerably weaker than the blue particles.The apparent quantum yield of the red particles was measured relative to4-(dicyanomethylene)-2-methyl-6-(4-dimethylamino-styryl)-4H-pyran (DCM)in methanol, with an assumed quantum yield of 0.38. For equivalentoptical densities at the excited wavelength of 442 nm, these particlesdisplay an apparent quantum yield of 0.015, and are thus less brightthan the blue-emitting CdS/dendrimer nanoparticles.

Compared to the blue-emitting nanoparticles, these red emittingparticles display simpler properties. The emission spectra areindependent of excitation wavelength, suggesting a narrow sizedistribution. The excitation spectrum (not shown) overlapped with theabsorption spectrum. These nanoparticles can be made to have a longwavelength absorption above 480 nm. The absorption and excitationspectra of the CdS/dendrimer particles also appeared to be identical(FIG. 1).

Excitation and emission anisotropy spectra of thesepolyphosphate-stabilized nanoparticles show zero anisotropy for allexcitation and emission wavelengths. The zero anisotropy values could bedue to rotational diffusion of the particles during these longluminescence decay (below).

However, time-dependent decay of the anisotropy is not detected, as seenfrom the frequency-domain anisotropy data. The nanoparticles in 80%glycerol at −60° C. also show the anisotropies to be zero for excitationfrom 350 to 475 nm (FIG. 8). These results suggest that polarizedemission is not a general property of nanoparticles, but requiresspecial conditions of synthesis or stabilizers.

The frequency-domain intensity decay of the PPS-stabilized nanoparticlesis shown in FIG. 9. The intensity decay is complex, again requiring atleast three decay times to fit the data (Table Ii). The intensity decayin the time domain is shown in FIG. 5. The decay times range from 150 nsto 25.3 μs, with a mean decay time near 9 μs. Once again there was aneffect of excitation wavelength, but less than seen with theblue-emitting CdS/dendrimer nanoparticles.

Observation of microsecond decay times for these red emitting particlesis an important result. There is currently considerable interest inusing red or near infrared (NIR) probes for non-invasive and/or in-vivomeasurements. Most such probes display relatively short decay times,typically less than 1 ns. While a few metal-ligand complexes are knownto emit in the red and to display long lifetimes the choice of probeswith long lifetimes are limited. These intensity decay data for thepolyphosphate-stabilized nanoparticles suggest that such nanoparticleprobes can provide a new class of luminophores with both long wavelengthemission and long decay times.

Commonly used quenchers sometimes do not affect nanoparticle emission.The effect of oxygen are shown in FIG. 10. Dissolved oxygen had a modesteffect on the intensity from the CdS/dendrimer particles, with theemission being quenched by about 40% for equilibration at one atmosphereof oxygen (top). Remarkably, dissolved oxygen had no effect on theemission from the CdS/PPS particles (lower panel). This is particularlysurprising given the long intensity decay time of these particles. Theabsence of quenching by oxygen could be a valuable result. For instance,the absence of oxygen quenching is a valuable property of thelanthanides, allowing long decay times in samples exposed to air. Theseresults suggest that some nanoparticles may be insensitive to oxygen,and thus useful for high sensitivity gated detection as is used in thelanthanide-based immunoassays. The CdS/dendrimer nanoparticles werequenched by both iodide and acrylamide (FIG. 11, top). The CdS/PPSparticles were quenched by iodide but not significantly by acrylamide(bottom). The quenching observed for both types of nanoparticles seemsto be at least partially dynamic, as seen by the decrease in mean decaytime (Table III).

Many potential applications of nanoparticles as luminescent probes areenvisioned. Red-NIR emitting probes with long decay times and optionallyresistance to oxygen quenching are envisioned. A favorable property ofthe nanoparticles is the long intensity decay times. This allows thoseparticles which display anisotropy to be used in hydrodynamic probes onthe timescales ranging from hundreds of nanoseconds to microseconds.This is a timescale not usually available to fluorescence without theuse of specialized luminophores. The luminescence decay times can beadjusted by changes in nanoparticles and nanoparticle composition,morphology, size, shape and surface modifications.

It is envisioned that the nanoparticles of the present invention coulddisplay resonance energy transfer. For example, the nanoparticles coulddisplay resonance energy transfer to absorbing dyes or could displayFörster transfer.

Sensors incorporating the nanoparticles of the present invention arealso envisioned for chemical, biological, optical and otherapplications. Preferred embodiments are sensors for important speciessuch as Ca²⁺, pH and/or chloride. Attachment of analyte-dependentabsorbers to the nanoparticles are envisioned for analyte-dependentemission.

Preferred methods of making the nanoparticles of the present inventionare described in Examples 1-2. Preferred methods of spectroscopicmeasurements of the nanoparticles of the present invention are describedin Example 3.

EXAMPLE 1 Nanofabrication of CdS/dendrimer Nanoparticles

The blue emitting CdS particles were prepared in the presence ofpoly(aminoamine) STARBURST® dendrimer, generation 4.0 (bow Corning,Midland, Mich.; Dendritech™, Inc., Midland, Mich.; Michigan MolecularInstitute, Midland, Mich.; Aldrich, Allentown, Pa.). The STARBURST®dendrimer (PAMAM) of generation 4.0 was purchased from Aldrich. Thisdendrimer is expected to have 64 surface amino groups. Based on themanufacturer's value of the dendrimer weight fractions in methanol, andthe known dendrimer densities, we prepared dendrimer stock solutions of1.14×10⁻⁴ M in methanol under a N₂ atmosphere at 10° C. The 2.0 mM stocksolutions of Cd²⁺ and S²⁻ were prepared by dissolving 62 mg ofCd(NO₃)2.4H₂O (Baker) in 100 mL of methanol, and by dissolving 15 mgNa₂S (Alfa) in 100 mL of methanol. The Cd²⁺ and S²⁻ stock solutions werefreshly prepared. In the standard incremental addition procedure, an0.50 mL aliquot of Cd²⁺ stock solution was added to 10 mL of thedendrimer stock solution at 10° C., followed by addition of an 0.50 mLaliquot of S²⁻ stock solution. The Cd²⁺ and S²⁻ additions were repeated10 times. The resulting solution was colorless and glowed bright blueunder UV Illumination. The product was stored in a freezer and did notshow any evidence of precipitation for months. This nanoparticledendrimer composite was stable for long periods of time in neutralmethanol.

EXAMPLE 2 Nanofabrication of CdS/PPS Nanoparticles

The red emitting particles are also composed of CdS, but stabilized withpolyphosphate [Mahtab, R., Rogers, J. P., and Murphy, C. J. (1995),Protein-sized quantum dot luminescence can distinguish between“straight”, “bent,” and “kinked” oligonucleotides, J. Am. Chem. Soc.117, 9099-9100]. For the polyphosphate-stabilized (PPS) CdS/PPSnanoparticles, 2×10⁻⁴ M Cd(NO₃)₂.4H₂O in degassed water was mixed withan equivalent amount of sodium polyphosphate, Na₆(PO₃)₆. Solid Na₂S wasadded, with vigorous stirring, to yield 2×10⁻⁴ M sulfide. The solutionimmediately turned yellow. Under UV light, the solution glowedred-orange.

EXAMPLE 3 Spectroscopic Measurements

Frequency-domain (FD) intensity and anisotropy decays were measured witha fluorescence spectrometer and standard fluorescence techniques [J. R.Lakowicz and I. Gryczynski, Topic in Fluorescence Spectroscopy, Vol I,Techniques, Plenum Press, New York, pp 293-355]. The excitation sourcewas a HeCd laser with an emission wavelength of 325 nm or 442 nm. Thecontinuous output of this laser was amplitude modulated with a Pockels'cell. The FD data were interpreted in terms of the multi-exponentialmodel: $\begin{matrix}{{l\quad (t)} = {\sum\limits_{i}\quad {\alpha_{i}\quad \exp \quad \left( {{- t}/\tau_{i}} \right)}}} & (1)\end{matrix}$

where α_(i) are the pre-exponential factors and τ_(i) are the decaytimes. The fractional contribution of each decay time component to thesteady state emission is given by $\begin{matrix}{f_{i} = {\left( {\alpha_{i}\tau_{i}} \right)/\left( {\sum\limits_{j}\quad {\alpha_{j}\tau_{j}}} \right)}} & (2)\end{matrix}$

Frequency-domain anisotropy decay data were measured and analyzed asdescribed previously [Lakowicz, J. R., Cherek, H., Kusba, J.,Gryczynski, I., and Johnson, M. L. (1993), Review of fluorescenceanisotropy decay analysis by frequency-domain fluorescence spectroscopy,J. Fluoresc. 3, 103-116] in terms of multiple correlation times:$\begin{matrix}{{r\quad (t)} = {\sum\limits_{k}\quad {r_{0k}\quad \exp \quad \left( {{- t}/\theta_{k}} \right)}}} & (3)\end{matrix}$

In this expression r_(0k) is the fractional anisotropy amplitude whichdecays with a correlation time θ_(k).

The foregoing examples are illustrative embodiments of the invention andare merely exemplary. A person skilled in the art may make variationsand modification without departing from the spirit and scope of theinvention. All such modifications and variations are intended to beincluded within the scope of the invention as described in thisspecification and the appended claims.

TABLE I Frequency-domain intensity and anisotropy decays of theCdS/dendrimer nanoparticles Exc. (nm) n^(a) τ (ns) α_(i) f_(i) X² _(R)395 1 61.8 1.0 1.0 1,136.9 2 6.2 0.747 0.137 116.0 0.253 0.863 32.0 33.1 0.748 0.090 50.2 0.163 0.319 169.8 0.089 0.591 1.1 325 1 52.3 1.01.0 991.5 2 7.8 0.705 0.160 97.9 0.295 0.890 37.5 3 2.7 0.699 0.080 39.50.205 0.341 142.8 0.096 0.579 1.7 ^(a)Number of exponents

At an excitation of 395 nm and an n^(a) of 1, the following anisotropydecay values are seen: θ_(k)=2,430.5 ns; r_(0k)=0.228; and X² _(R)=0.6

TABLE II Frequency-domain intensity decay of the Cd²⁺ enrichednanoparticles Exc. (nm) n^(a) τ (ns) α_(i) f_(i) X² _(R) 442 1 597.501.00 1.00 1,656 2 290.40 0.932 0.448 4,907 0.068 0.552 242.90 3 150.000.749 0.188 1,171 0.243 0.476 25,320 0.008 0.336 2.70 325 1 680.20 1.001.00 1212.30 2 425.00 0.932 0.474 6,471 0.068 0.526 93.50 3 241.60 0.7170.227 1,173 0.273 0.421 27,783 0.010 0.352 2.90 ^(a)Number of exponents

TABLE III Intensity decay of the nanoparticles with and withoutquenchers. τ(avgas) τ₁ τ₂ τ₃ Compound/Conditions (ns) α₁ (ns) α₂ (ns) α₃(ns) X² _(R) blue, no quencher 106.0 0.698 4.91 0.256 57.7 0.046 214.22.2 blue + 0.2 M acrylamide 73.7 0.737 1.07 0.190 18.0 0.073 105.9 4.2blue + 0.2 M iodide 36.7 0.786 1.11 0.175 11.2 0.039 67.3 4.6 red, noquencher 9.80 0.652 232.5 0.337 1073.3 0.011 2580 3.8 red + 0.2 Macrylamide 8.54 0.761 229.3 0.229 1173.0 0.010 2349 1.9 red + 0.2 Miodide 4.09 0.738 56.3 0.243 673.7 0.019 858.2 3.2 ^(b)The excitationwas 325 nm. The emission filter for the blue particles was aninterference filter 500 +/− 20 nm. The emission filter for the redparticles was a long pass filter at 580 nm.${\quad^{c}{\tau ({avgas})} = {\sum\limits_{i}^{\quad}\quad {f_{i}\quad {\tau ({avgas})}i}}};$

$f_{i} = {\alpha_{i}{\tau_{i}/\left( {\sum\limits_{i}^{\quad}\quad {\alpha_{j}\tau_{j}}} \right)}}$

We claim:
 1. A fluorescent nanoparticle comprising cadmium sulfide; andat least one dendrimer.
 2. A fluorescent nanoparticle of claim 1 whereinsaid at least one dendrimer is selected from the group consisting ofamine-terminated dendrimer, poly(amidoamine) dendrimer,poly(amidoamino-organosilicon) dendrimer having hydrophilic domains andhydrophobic domains, star polymers, self-assemblying polymers, andzeolites.
 3. A fluorescent powder comprising: a plurality of thefluorescent nanoparticles of claim 1 wherein said fluorescentnanoparticles have an average critical dimension ranging fromapproximately 2 nm to approximately 5 nm and wherein the majority of theplurality of said fluorescent nanoparticles have a size distributionwithin approximately +/−15% of the average critical dimension.
 4. Afluorescent power of claim 3 wherein said at least one dendrimer isselected from the group comprising amine-terminated dendrimer,poly(amidoamine) dendrimer, poly(amidoamino-organosilicon) dendrimerhaving hydrophilic domains and hydrophobic domains, star polymers,self-assemblying polymers, and zeolites.
 5. A process for making thefluorescent nanoparticle of claim 1 comprising mixing a Cd(NO₃)₂.4H₂Omethanol solution with a molar equivalent methanol solution ofdendrimer; and adding Na₂S while in deoxygenated environment.
 6. Thefluorescent nanoparticle of claim 1, wherein the fluorescentnanoparticle has a wavelength emission of at least 500 nm and a decaylifetime of at least 30 ns.
 7. A sensor for the measuring of theconcentration of an analyte or the absence of an analyte in a sample,said sensor comprising: a signal emitting probe further comprising afluorescent nanoparticle of claim 1; a light source; a light filter; afluorescent light detector; a fluorescence measurement system; and adata analysis system wherein said data analysis system converts themeasured fluorescence of a signal to a concentration for an analyte. 8.A fluorescent nanoparticle comprising cadmium sulfide; andpolyphosphate.
 9. A fluorescent powder comprising: a plurality of thefluorescent nanoparticles of claim 8 wherein said fluorescentnanoparticles have an average critical dimension ranging fromapproximately 2 nm to approximately 5 nm and wherein the majority of theplurality of said fluorescent nanoparticles have a size distributionwithin approximately +/−15% of the average critical dimension.
 10. Aprocess for making a fluorescent nanoparticle of claim 8 comprising:mixing a Cd(NO₃)₂.4H₂O methanol solution with a molar equivalentmethanol solution of Na₆(PO₃)₆; and adding Na₂S while in deoxygenatedenvironment.
 11. The fluorescent nanoparticle of claim 8, wherein thefluorescent nanoparticle has a wavelength emission of at least 500 nmand a decay lifetime of at least 30 ns.
 12. A sensor for the measuringof the concentration of an analyte or the absence of an analyte in asample, said sensor comprising: a signal emitting probe furthercomprising a fluorescent nanoparticle of claim 8; a light source; alight filter; a fluorescent light detector; a fluorescence measurementsystem; and a data analysis system wherein said data analysis systemconverts the measured fluorescence of a signal to a concentration for ananalyte.